Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.     

Muscle cellular properties in the ice hockey player: a model for investigating overtraining?

In this study, we hypothesized that athletes involved in 5-6 months of sprint-type training would display higher levels of proteins and processes involved in muscle energy supply and utilization. Tissue was sampled from the vastus lateralis of 13 elite ice hockey players (peak oxygen consumption = 51.8 ± 1.3 mL·kg(-1)·min(-1); mean ± standard error) at the end of a season (POST) and compared with samples from 8 controls (peak oxygen consumption = 45.5 ± 1.4 mL·kg(-1)·min(-1)) (CON). Compared with CON, higher activities were observed in POST (p < 0.05) only for succinic dehydrogenase (3.32 ± 0.16 mol·(mg protein)(-1)·min(-1) vs. 4.10 ± 0.11 mol·(mg protein)(-1)·min(-1)) and hexokinase (0.73 ± 0.05 mol·(mg protein)(-1)·min(-1) vs. 0.90 ± 0.05mol·(mg protein)(-1)·min(-1)) but not for phosphorylase, phosphofructokinase, and creatine phosphokinase. No differences were found in Na(+),K(+)-ATPase concentration (β(max): 262 ± 36 pmol·(g wet weight)(-1) vs. 275 ± 27 pmol·(g wet weight)(-1)) and the maximal activity of the sarcoplasmic reticulum Ca(2+)-ATPase (98.1 ± 6.1 μmol·(g protein)(-1)·min(-1) vs. 102 ± 3.3 μmol·(g protein)(-1)·min(-1)). Cross-sectional area was lower (p < 0.05) in POST but only for the type IIA fibres (6312 ± 684 μm(2) vs. 5512 ± 335 μm(2)), while the number of capillary counts per fibre and the capillary to fibre area ratio were generally higher (p < 0.05). These findings suggest that elite trained ice hockey players display elevations only in support of glucose-based aerobic metabolism that occur in the absence of alterations in excitation-contraction processes.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.     

Molecular cloning of a mitogenic proteinase from Carica candamarcensis: its potential use in wound healing

Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment.     

不同光照条件下外源水杨酸对浒苔响应紫外辐射胁迫的影响

为研究不同光照条件下,外源水杨酸(SA)和紫外辐射(UV)对海洋绿藻浒苔的复合效应,在两个光照强度(高光:160 μmol·m^-2·s^-1;低光:70 μmol·m^-2·s^-1)条件下,设置对照(CK)、SA、UV及UV+SA处理(UV=3.2 W·m^-2、SA=10 μg·mL^-1),处理3 d后测定浒苔生长、叶绿素荧光参数、光合放氧速率、超氧化物歧化酶活性、可溶性糖和可溶性蛋白含量等的变化,探讨光照强度、UV及SA的复合效应.结果表明: 低光无UV条件下,SA会促进浒苔生长,降低浒苔叶绿素a(Chl a)和可溶性蛋白含量;高光无UV条件下, SA会抑制其生长,但显著提高了Chl a含量、呼吸速率、光合放氧速率、可溶性糖和可溶性蛋白含量;高光和UV条件下, UV+SA显著促进浒苔生长,提高Chl a和可溶性糖含量;低光和UV条件下,与UV相比,UV+SA提高了浒苔最大光化学效率(F_v/F_m)和可溶性蛋白含量,涨幅分别为139.8%和32.2%.外源SA的加入在一定程度上缓解了UV对浒苔的胁迫作用,且在高光条件下的效果更为显著.     

Identification of a new phospholipase D in Carica papaya latex

Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform-methanol/TCA-acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family.     

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

Isolation and Characterization of β-1,4- Endoxylanase from Trichoderma pseudokonigi

13-1,4-endoxylanase from Triehoderma pseudokonigi Rifai has been purified by anion-exchange chromatography on DEAE-Sephadex A50, DEAE-Sepharose CL-6B and mono Q. The endoxylanase was shown to be homogeneous by Native-PAGE and SDS-PAGE. This endoxylanase is a single-peptide chain protein with a molecular weight estimated as 66 kD. The endoxylanase was purified by 10-fold with a specific activity of 15.87 U·mg－1 Optimum endoxylanase activity was obtained when the enzyme was incubated at pH 4.5, 55 ℃ with a Km of 20 mg/mL and Vmax of 3.3 μmol·min－1·mg－1. Hg2 + and Cu2 + have a strong inhibition while Fe2 + and Mn2 + have a increasing effect on the enzymatic reaction rate.     

华北大黑鳃金龟中肠丝氨酸蛋白酶cDNA克隆、 序列分析及表达

丝氨酸蛋白酶是昆虫体内一类重要的消化酶, 为了了解该类酶的分子特性及功能, 本研究利用粉纹夜蛾Trichoplusia ni围食膜蛋白多克隆抗体筛选华北大黑鳃金龟Holotrichia oblita中肠cDNA表达文库, 首次得到编码华北大黑鳃金龟丝氨酸蛋白酶cDNA序列, 命名为HoSP1（GenBank登录号为FJ573146）。序列分析表明, 该基因长902 bp, 开放阅读框（ORF）长783 bp, 编码260个氨基酸, 推测分子量和pI值分别为26.7 kDa和4.19, 不含有N-糖基化位点, 但在Thr₁₅₇处有一个O-糖基化位点, 含有6个保守的半胱氨酸残基, 组成3对二硫键, 对于维持蛋白质的三级结构起着重要的作用。通过与几种丝氨酸蛋白酶的比对发现, 该酶具有组氨酸（His）、 天冬氨酸（Asp）、 丝氨酸（Ser）催化中心, 与褐新西兰肋翅鳃金龟Costelytra zealandica的14种丝氨酸蛋白酶有明显的相似性, 其中与CzSP3的序列一致性最高, 为52.47%。把该基因与pET21b载体重组后, 进行体外表达, 以BTEE为底物, 测得该酶的活力为0.0378 μmol/mg·min。HoSP1基因的克隆及体外表达为进一步研究该酶在华北大黑鳃金龟体内的表达及功能提供了依据。     

Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

A novel extra-cellular lipase from Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103 kDa. The lipase was optimally active at 45 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was more specific to p-nitrophenyl esters of 8 (pNPC) and 16 (pNPP) carbon chain length esters. The lipase had a Vmax and Km of 0.44 mmol mg(-1)min(-1) and 28 mM for hydrolysis of pNPP, and 0.7 mmol mg(-1)min(-1) and 32 mM for hydrolysis of pNPC, respectively.     

Characterization of the proteolytic system present in Vasconcellea quercifolia latex

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia (“oak leaved papaya”) latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman’s degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.     

Prolonged infusion of amino acids increases leucine oxidation in fetal sheep

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ～12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.     

The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2)

During the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two approximately 10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium-independent phospholipases A(2). Detailed inspection of the identified sequence (residues 79, 671-109,912 GenBank accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A(2) through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5'-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10-fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0. 3 nmol/mg.min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC(50) = 3 microM), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, and sn-2-lipase activity of a novel intracellular calcium-independent membrane-associated phospholipase A(2).     

Glutamine and α-ketoglutarate as glutamate sources for glutathione synthesis in human erythrocytes

Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, α-ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mm extracellular α-ketoglutarate, but not 10 mm glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 μmol·(L RBC)(-1) ·min(-1), respectively) compared with 0.52 ± 0.09 μmol·(L RBC)(-1) ·min(-1) for RBCs without an external glutamate source. Mathematical modelling of the situation with 0.8 mm extracellular glutamine returned a rate of glutamate production of 0.36 μmol·(L RBC)(-1) ·min(-1), while the initial rate for 0.8 mM α-ketoglutarate was 0.97 μmol·(L RBC)(-1) ·min(-1). However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with α-ketoglutarate (0.31 and 0.25?μmol·(L RBC)(-1) ·min(-1), respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of α-ketoglutarate and/or glutamine.     

Purification and characterization of lipases from wheat germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivum) is described. Electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 x x 10(-3) mumol/min per mg protein) was obtained after purification in 61 times. The molecular weight of the enzyme, determined by gel chromatography, was 143 +/- 2 kDa. The optimal conditions for the enzyme were 37 degrees and pH 8.0. Homogeneous preparation of the lipase exhibited high thermal stability: over 20% of original activity was retained after incubation of the preparation at high temperatures (60-90 degrees) for 1 h at pH 8.0.     

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(?)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.     

镉对小鼠精子蛋白酪氨酸磷酸化修饰的影响及EGTA的保护作用

采用体外培养的方法,利用精子活力分析软件(CASA)、蛋白免疫印迹(WB)及免疫荧光技术, 探讨镉(Cd)对小鼠精子活力参数、蛋白酪氨酸磷酸化修饰的影响,并对小鼠精子酪氨酸磷酸化蛋白进行细胞亚组分定位. 结果表明: Cd对小鼠精子活力具有明显抑制作用,且随着Cd浓度的增加抑制作用增强,当Cd浓度达到1.0 μmol·L^-1时, 小鼠精子活力(MOT)显著低于对照组;同时,Cd促进小鼠精子蛋白酪氨酸磷酸化,当浓度≥1.0 μmol·L^-1时,尤其分子量约为55 kDa的蛋白酪氨酸磷酸化程度显著增强,且免疫荧光结果显示主要集中在小鼠精子中段;当用30 μmol·L^-1 乙二醇二乙醚二胺四乙酸(EGTA)和10 μmol·L^-1 Cd同时培养时,55 kDa蛋白并未发生明显的酪氨酸磷酸化修饰,而且小鼠精子活力变化不显著. 表明Cd可能是通过诱导中段55 kDa蛋白发生酪氨酸磷酸化修饰从而抑制精子活力,EGTA能螯合Cd离子并有效防止其毒性作用. 研究证实,Cd诱导精子特异性蛋白酪氨酸磷酸化增强,进而抑制精子活力. EGTA可以用于体外控制Cd进入细胞的阻断剂,为Cd繁殖毒性分子机制的研究提供了研究手段.     

Novel soluble epoxide hydrolase inhibitor protects mitochondrial function following stress

Epoxyeicosatrienoic acids (EETs) are active metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase enzyme (sEH) to dihydroxyeicosatrienoic acid. EETs are known to render cardioprotection against ischemia reperfusion (IR) injury by maintaining mitochondrial function. We investigated the effect of a novel sEH inhibitor (sEHi) in limiting IR injury. Mouse hearts were perfused in Langendorff mode for 40 min and subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with 0.0, 0.1, 1.0 and 10.0 μmol·L(-1) of the sEHi N-(2-chloro-4-methanesulfonyl-benzyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide (BI00611953). Inhibition of sEH by BI00611953 significantly improved postischemic left-ventricular-developed pressure and reduced infarct size following IR compared with control hearts, and similar to hearts perfused with 11,12-EETs (1 μmol·L(-1)) and sEH(-/-) mice. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol·L(-1)), or the plasma membrane K(ATP) channels (pmK(ATP)) inhibitor (glibenclamide, 10 μmol·L(-1)) abolished the improved recovery by BI00611953 (1 μmol·L(-1)). Mechanistic studies in H9c2 cells demonstrated that BI0611953 decreased ROS generation, caspase-3 activity, proteasome activity, increased HIF-1∝ DNA binding, and delayed the loss of mitochondrial membrane potential (ΔΨ(m)) caused by anoxia-reoxygenation. Together, our data demonstrate that the novel sEHi BI00611953, a nicotinamide-based compound, provides significant cardioprotection against ischemia reperfusion injury.     

Effects of raising muscle glycogen synthesis rate on skeletal muscle ATP turnover rate in type 2 diabetes

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. We hypothesized that any impairment in insulin-stimulated muscle ATP production could merely reflect the lower rates of muscle glucose uptake and glycogen synthesis, rather than cause it. If this is correct, muscle ATP turnover rates in type 2 diabetes could be increased if glycogen synthesis rates were normalized by the mass-action effect of hyperglycemia. Isoglycemic- and hyperglycemic-hyperinsulinemic clamps were performed on type 2 diabetic subjects and matched controls, with muscle ATP turnover and glycogen synthesis rates measured using (31)P- and (13)C-magnetic resonance spectroscopy, respectively. In diabetic subjects, hyperglycemia increased muscle glycogen synthesis rates to the level observed in controls at isoglycemia [from 19 ± 9 to 41 ± 12 μmol·l(-1)·min(-1) (P = 0.012) vs. 40 ± 7 μmol·l(-1)·min(-1) in controls]. This was accompanied by a modest increase in muscle ATP turnover rates (7.1 ± 0.5 vs. 8.6 ± 0.7 μmol·l(-1)·min(-1), P = 0.04). In controls, hyperglycemia brought about a 2.5-fold increase in glycogen synthesis rates (100 ± 24 vs. 40 ± 7 μmol·l(-1)·min(-1), P = 0.028) and a 23% increase in ATP turnover rates (8.1 ± 0.9 vs. 10.0 ± 0.9 μmol·l(-1)·min(-1), P = 0.025) from basal state. Muscle ATP turnover rates correlated positively with glycogen synthesis rates (r(s) = 0.46, P = 0.005). Changing the rate of muscle glucose metabolism in type 2 diabetic subjects alters demand for ATP synthesis at rest. In type 2 diabetes, skeletal muscle ATP turnover rates reflect the rate of glucose uptake and glycogen synthesis, rather than any primary mitochondrial defect.